1. Field of the Invention
The present invention relates to biochemical procedures and, in particular, to a method for rapidly differentiating gram-positive and gram-negative microorganisms.
2. Description of Related Art
All microorganisms can be separated into one of two classes, either gram-positive or gram-negative, based on their gram-stain reaction. The gram reaction is, therefore, a key test in the identification of microorganisms. In addition, owing to general structural and biochemical differences between gram-positive and gram-negative microorganisms, a different spectrum of antibiotics is used to treat infections caused by one of these classes than is used to treat infections caused by the other. Knowledge of the gram reaction of an infecting organism is, therefore, important for immediate selection of an appropriate antibiotic.
Currently, the gram-stain is a four-step staining procedure performed on a glass slide containing dried heat-fixed biological material. The method as it is commonly used today is the Hucker modification of the original staining procedure developed in 1883 by Hans Christian Gram.
To perform a gram stain, the slide is first flooded with a primary stain, a basic dye such as crystal violet, followed by the addition of 2-3 drops of a sodium bicarbonate solution to provide the necessary alkaline conditions. Alternatively, the sodium bicarbonate may be added directly to the crystal violet solution. After one minute, the crystal violet is rinsed off with tap water and is followed by the addition of a mordant, an iodine-potassium iodide mixture, which complexes with the crystal violet. The iodine is left on the slide for one minute and then gently washed off with tap water.
Decolorization to remove excess dye and dye from gram-negative cells is accomplished by allowing either a mixture of acetone and alcohol or an aqueous alcohol solution to flow down the slide until the runoff is colorless. This step is critical and either over or under decolorization can cause false results.
After rinsing with water, a counterstain is applied for one minute. The slide is again rinsed with water, air dried or blotted dry with absorbent paper, and examined visually under an oil immersion lens. Gram-positive organisms will retain the primary stain, while gram-negative organisms will be stained with the counterstain. Bartholomew et al., "The Gram Stain", Bact. Rev., 16:1-29, 1952 is a comprehensive review article describing this procedure and its interpretation. In Table 1, on page 10, of this reference, it is pointed out that gram-positive cells are more susceptible to death or growth inhibition by anionic detergents in general and higher alkyl sulfates. However, as shown in Example 5 below, compounds effective in growth inhibition are not necessarily effective in selectively inhibiting dye reduction in the rapid assay described herein.
Owing to the number of steps involved and the importance of proper decolorization technique, which will vary with type and amount of material present on the slide, as well as the need for heat-fixing the specimen to the slide, the actual staining procedure is difficult to automate completely. Furthermore, the entire procedure, including preparation of the slide, staining and viewing is time consuming, especially when numerous samples are involved. A one-step determination of the gram reaction that could be completely automated would greatly expedite this procedure and would therefore find a useful position in clinical microbiology.
U.S. Pat. No. 4,225,669 describes a bacterial staining composition and methods for analysis of both gram-negative and gram-positive bacteria. The staining composition comprises a chelating agent and a basic dye, both of which are operative at a pH above 7.0. Bacteria thus stained and concentrated by filtration or centrifugation are readily visible and semi-quantitative analysis is accomplished by comparing the gradation of color developed with a calibrated standard.
Differentiation of gram-negative and gram-positive bacteria is accomplished by treating the stained bacteria with an organic acid wash having a pH of about 2.5 to 2.6, which completely decolorizes only the gram-positive bacteria and thereby differentiates them from the gram-negative bacteria.
Chapman, "A Superior Culture Medium for the Enumeration and Differentiation of Coliforms," J. Bact., 53:504, 1947, describes a culture medium containing a surfactant, Tergitol-7, which inhibits or limits growth of non-coliform bacteria.
Pollard, "A Useful Selective Bactericidal Property of Tergitol-7," Science, 103:758-759, 1946, discusses the selective growth inhibition of a heat resistant bacillus in a starch/tryptone/agar medium by Tergitol-7 at a final concentration of 1:20,000 (Tergitol-7:bacillus).
Baker et al., "Action of Synthetic Detergents on the Metabolism of Bacteria," J. Exp. Med., 73:249-271, 1941, correlates chemical structure and properties of synthetic detergents with their effects on bacterial metabolism as determined by respiratory techniques. These effects were evaluated based on oxygen uptake by the bacteria. Anionic detergents, e.g., Tergitol-7, inhibited gram-positive bacterial respiration maximally at an acid pH and at a concentration of 1:3,000. Anionic detergents inhibited only the metabolism of gram-positive microorganisms, whereas cationic detergents inhibited the metabolism of both gram-positive and gram-negative microorganisms to the same degree. Tergitol-7 inhibited one gram-negative microorganism, Proteus vulgaris. Baker et al. observe on page 261 of the reference that the anionic detergents have the capacity to differentiate sharply between gram-positive and gram-negative microorganisms. However, this observation was based solely on respirometric procedures and would in no way suggest to a person of ordinary skill in the art a means for protecting a reducible compound in the presence of a gram-positive microorganism or a practical and rapid method for gram separation. Furthermore, the method would be expected to be limited to reactions occurring under aerobic conditions.
Dakay et al., "The Effect of Synthetic Detergents on Formazan Formation of Various Environmental Bacteria", Zentralblatt Bakt. Hyg. I Abt. Orig. B 174, pages 121-4 (1981) studied the effect of five detergents on dehydrogenase activity of a few bacteria as an indicator of environmental pollution. Two gram-positive organisms and two strains of a single gram-negative organism were used. Triphenyl tetrazolium chloride was used as an indicator of dehydrogenase activity. The cationic surfactant tested was very inhibitory to dehydrogenase activity. Of the two anionic detergents tested, the inhibitory effect was most noticeable with Streptococcus faecalis (0.03-0.08%), with Micrococcus Sp. (another gram-positive organism) proving to be less sensitive. E. coli (a gram-negative organism was inhibited at much higher concentrations. Non-ionic surfactants had no inhibitory effects.